Stimulation of the Human Intercellular Adhesion Molecule-1 Promoter ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269,No. 33, Issue of August 19,PP. 2114621154, 1994 Printed in U.S.A.

Stimulation of the Human Intercellular Adhesion Molecule-1 Promoter by Interleukin-6 and Interferon-y Involves Bindingof Distinct Factors to a Palindromic Response Element* (Received forpublication, January 31, 1994, and in revised form, May 19, 1994)

Eric CaldenhovenSPn, Paul CofferSPII, Juping Yuan**, Anja Van De StolpeS, Friedemann Horn**, Wiebe KruijerS, and PaulT.Van Der SaagS From the Wubrecht Laboratorium, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands, the **Znstitute for Biochemistry, Pauwelstrasse 30, 0-52057 Aachen, Federal Republic of Germany, and the lDepartment of Pulmonary Diseases, University Hospital Utrecht, I? 0.Box 85500 Utrecht, The Netherlands

Intercellular adhesion molecule-1(ICAM-1)is a transmembrane glycoprotein that promotes adhesion in immunological and inflammatory reactions.ICA"1 is expressed on cells of many lineages and is induced by interleukin-6 (IL-6)and interferon-y (IFN-y). Functional analysis of ICA"1 promoter-luciferase constructs in HepG2 cells enabledus to identify a region between-110 and -37 mediating IL-6 and IFN-y responsiveness and containing a palindromic IL-6/IFN-y response element (PIRE). Site-directed mutagenesis of key nucleotides in the I C A " 1 pIRE abolished the effect of both IL-6 and IFN-y stimulation, while this pIRE element was sufficient to confer IL-6 and IFN-y responsiveness to a heterologous promoter.We further show by gelretardation analysis that distinct nuclear factors induced by both IL-6 or IFN-y specifically bind to this pIRE. Furthermore, treatment with IL-6 results in the formation of IFN-y induces a single binding multiple complexes while complex, both in HepG2 and monocytic U937 cells. Differentiation of U937 cells by exposure to 12-0-tetradecanoyl phorbol-13-acetate abolishes response to IL-6 but not IFN-y. Supershift data utilizing the ICA"1 pIRE revealed that IFN-y and IL-6 both induce a factor antigenically related to IFN-y activation factor. We further provide data suggesting that IL-6 additionally activates an ICA"1 pIRE binding factor related to the previously described acute-phase response factor in disparate cell types. We therefore conclude that theactivation of these related nuclear factors by IL-6and IFN-y is important in the regulation of ICA"1 gene expression.

inflammatory responseICA"1 is expressedon a limited number of cell types, but expression can be induced and up-regulated in various cell lineages of both hemopoietic and nonhemopoietic origin by inflammatory mediators such as tumor necrosis factor a (TNF-a), interleukin-6(IL-6), and interferon-? (IFN-y) (4-9). The regulation of ICA"1 expression by these cytokines is essential for cell-mediated cytotoxicity and for leukocyte migration to sitesof inflammation (1, 10). TNF-a, IL-6, and IFN-y act by binding tospecific receptors on the cell membrane (11-13), therebyinitiatingsignaltransductionpathways, which finally leads to induction of specific gene expression. The 5"flanking region of the ICA"1 gene contains all the necessaryelements for induction of I C A " 1 expression by theseinflammatory cytokines (14-17). Although IL-6 and IFN-y are different typesof cytokines they havesome overlapping biological activities. For example, with respect t o gene induction, they both increase the level of interferon regulatory factor 1(IRF-1) (18, 191, and the acute-phase protein C1 inhibitor (86). Moreover, it has been shownthat both cytokines can up-regulate I C A " 1 mRNA levels (4, 5, 10, 201.' IL-6 is a pleiotropic cytokine which is involved in immune and inflammatory responses(21). It stimulatesB-cell differentiation and IgG secretion (22), induces differentiation of myeloid cells (231, and stimulates the production of acute-phase proteins by the liver (24). IL-6 exerts these multipleeffects by binding t o an 80-kDa IL-6 receptor subunit (25) which upon activation associates with the signal transducing receptor component gp130 (13, 26) activating anintracellularsignaling pathway which is initiated by tyrosine phosphorylation (20,27, 28). Nuclear transcription factors activated by the IL-6 response mediate their effects through binding cis-acting eleIntercellular adhesion molecule 1 (ICAM-1)' is a n inducible ments in the promoters of target genes(29-31) and a variety of cell surface glycoprotein that promotes leukocyte adhesion and enhancer elements regulating the response of promoters have migration in immunological and inflammatory reactions (1). been identified (32-36). Interferon-y (IFN-y), a cytokine proICA"1 is an adhesive ligand for the leukocyte integrins LFA-1 duced by activated T lymphocytes,is a potent activatorfor cells (CDlldCD18) (2) and Mac-1 (CDllb/CDlS) (3). Prior t o an of the mononuclear phagocyte lineage, and therefore plays a critical role in host defense and inflammation (37, 38). It in* The costsof publication of this article were defrayed in part by the duces expressionof cell-surface molecules, including classI and payment of page charges. This article must therefore be hereby marked class I1 antigens of the major histocompatibility complex (39, "advertisement"in accordance with 18 U.S.C.Section 1734 solelyto 40) and Fc receptors (41, 42). IFN-y binds to high affinity reindicate this fact. ceptors on the cell surface (ll),thereby activating the DNA5 The first two authors contributed equally. 11 To whom corresponding should be addressed: Hubrecht Laborato- binding protein y-interferon activation factor (GAF) (43). In rium, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Tel.: 31-30- response to IFN-y, this 91-kDa protein is rapidly phosphoryl510211; Fax: 31-30-516464. ated on tyrosine (44, 45) and is translocated to the nucleus The abbreviations used are: ICA"1, intercellular adhesion mole- where it binds to a DNA element, the y-activated site (GAS) cule-1;IL-6,interleukin-6;IFN-y, interferon gamma; pIRE,palin(43, 46). To investigate the mechanisms by whichIL-6 and dromic IL-6/IFN-y response element; GAF, IFN-yactivationfactor; effects in regulating expression of the human APRF,acute-phase response factor; TNF-a,tumor necrosis factor-a; IFN-y exert their IRF-1, interferonregulatory factor-1; APRE, acute-phase response element; luc, luciferase; TPA, 12-0-tetradecanoyl phorbol-13-acetate; GAS, * E. Caldenhoven, andP. Coffer, unpublished results. IFN-y activation site; bp, base pairk).

21146

ICAM-1 Promoter Regulation by IL-6 and IFN-y I C A " 1 gene, we have performed a functional analysis of the 5"flanking region by transiently transfecting reporter constructs in HepG2 cells. Our findings identified an IL-6 and IFN-y-responsive region between the nucleotides -76 and -66 which we designated a palindromic IL-6IIFN-y response element (pIRE). We further analyzed the effects of IL-6 and IFN-y on ICAM-1 pIRE binding ina gel retardation assay andfound that IL-6 and IFN-y induce nuclear factors that specifically bind to thisDNA motif. Furthermore, we provide evidence that both IL-6 and IFN-y induce a GAF-related complex, whereas IL-6 induces also a second nuclear factor which has similar characteristics to thatpreviously described for AF'RF (36). Finally, we show that these nuclear factors are also induced by IL-6 and IFN-7 in the monocytic cell line U937 and thus are likely candidates for a role in ICA"1 expression in thesecells.

21147

final volume of 20 pl, containing 10mM HEPES, pH 7.8, 50 mM KCI, 1 mM EDTA, 5 m~ MgCl,, 10% (v/v) glycerol, 5 m~ dithiothreitol, 2 pg poly(d1-dC)(Pharmacia), and 20 pg bovine serum albumin with0.1-1.0 ng of 32P-labeled 1CA"pIRE oligonucleotide for 20 min a t room temperature. In competition analysis, extracts were incubated for 5 min with the indicated molar excess ofcold oligonucleotide prior to the addition of the labeled oligonucleotides. Supershift analysis were performed by preincubating nuclear extracts from HepG2 cells treated for 15 min with IL-6 or IFN-y,for 30 min with 1:lOO dilution of the antip91, anti-p91/84, and anti-pll3 before the addition of the probe. Subsequently, samples were run for 2 h (3-4 h fora long-run gel) on a5% non-denaturing polyacrylamide gel at room temperature and f=ed in 10% (vh) methanol/lO% (v/v) aceticacid,vacuum-dried,andfinally exposed to Fuji RX film at -70 "C for 1-2 days. RESULTS

Functional Analysis of the Z C M - 1 Promoter, Identification of a n ZL-6- and IFN-y-responsive Region-Analysis of the 5'MATERIALSAND METHODS flanking region of the human ICA"1promoter reveals anumCell CuZture, Reagents, a n d Antibodies-The human hepatoma cell ber of putative cis-acting elements implicated in theregulation line, HepG2, was grown in 1:l mixture of Dulbecco's modified Eagle's of eukaryotic genes. Among them are binding sites for SP1, medium and Ham's F-12 medium buffered with bicarbonate and suppleAP2, AF'1, and NFKB(Fig. lA).It hasrecently been shown that mented with 10% heat-inactivated fetal calf serum. The promonocytic I C A " 1 gene expression is induced by IL-6 and IFN-y in sevU937 cell line is a human histiocytic lymphoma cell line and possesses eral cell types (4-7, 20).3 To study the mechanisms by which characteristics of monocytic cells. U937 cells (ATCC CRL 1593, Rockville, MD) were cultured in RPMI 1640 medium containing 10% fetal these cytokines activate the ICA"1promoter, we generated a calf serum and kept at a density below 5 x lo5 cells/ml. For differen- series of 5' and internal promoterdeletions constructs and tiation U937 cells were transferred to Petri dishes at a concentration of cloned these ina promoterless luciferasevector p X P 2 (Fig. 1B1. 2 x lo6 celldm1 and treated with a final concentration of 16 nM 12-0- We used these promoter-luciferase constructsintransient tetradecanoylphorbol-13-acetate(TPA, Sigma) for 48 h. For cytokine transfections of the human hepatoma cell line HepG2. These treatment, cells were exposed to human recombinant IL-6, 100 BSF-2 cells were utilized for this study as theyrespond well to both (B-cell stimulatory factor2) unitdml, or human recombinant IFN-y 100 IL-6 and IFN-y and are easily transfected. After a 24-h period unitdml (both from Boehringer). The antisera against the ISGF3a subunits, the 91-kDa (anti-pglc), the 91/84-kDa (anti-p84/91), and the113- of expression of the transfectedDNA, cells were treated for 16 kDa (anti-pll3) were a generous gift from C. Schindler, New York and h with IL-6 or IFN-y and the luciferase activity measured. have been described previously (47,48), while p9W84-specific a antisera Promoter activity was also measured after4- and 8-h periodsof from Transduction Labs (Kentucky) was alsoemployed. cytokine stimulation with similar results. Treatment with IL-6 Synthetic Oligonucleotides, Plasmid Construction, and Mutaof cells transfected with construct PIC-1014 gives a 6-fold ingenesis-Oligonucleotides with the following sequences were synthesized (only the top strands are shown). The human ICA"1pIRE(1C): duction compared with untreated cells (Fig. =), while stimulation of this construct withIFN-y revealed a lower %fold in5'-agcttAGG'ITTCCGGGAAAGCAc-3', contains a t both sites HindIII linkers; human ICA"1 pIRE(m): 5"AGGCGCGAGGTTAGCGGTduction. A5' deletion up to-277 resulted in retained sensitivity CAAGCAGCACCGC-3'; rat a,-macroglobulin APRE "core": B'GATCCTt o both stimuli, and the removal of the nucleotides -227 to -136 TCTGGGAATTCCTA-3' (49); rat a,-macroglobulin APRE "core" from construct PIC-277 also had no effect on the induction by mutant: 5'-GATCCTTCTCTAGATTCCTA-3'(36). Double-stranded oligonucleotides were preparedby annealing the complementary single IL-6 or IFN-y. However, deletion of the region -105 to -37 from strands. The ICA"1 promoterdeletion constructs PIC-1014, PIC-677, either constructPIC-277 or PIC-135 resulted ina complete loss PIC-339, PIC-277, PIC-277AAP2, pIC-277ASMA, PIC-135, PIC- of response to IL-6 or IFN-y (Fig. 1B).Deleting this region also 135AAP2, PIC-34, and the luciferase vector pXP2 were previously de- reduced basal activity of the ICA"1 promoter. This indicates scribed (17). Thel x and 2x pIREtkluc were constructed by cloning the that an IL-6- and/or IFN-y-inducible response element(s) is oligonucleotide pIRE(1C) in the HindIII site in front of the herpes virus located between the nucleotides -105 and -37 of the human thymidine kinase minimal promoter in the pl9tklucvector (50), while I C A " 1 promoter. the pSV-lacZ construct has been previously described (51). The pIRE A pZRE in the ICA"1 Promoter Is Responsive to Both IL-6 mutation in the ICA"1 promoter was created using the Promega Altered Sites kit. A 1014-bp BarnHIISstI fragment of PIC-1014 was sub- and ZFN-y-Analysis of the region -105 to -37 of the ICAM-1 cloned into the pSelect vector and used as a template for the in vitro sequence 5'-l"TCCGGpromoter reveals a palindromic synthesis of the complementary strand. Mutant clones were sequencedGAAA-3' between nucleotides -76 to -66. To examine whether and the 1014-bp BarnHIISstI fragment was excised and recloned into this elementis involved in mediating the stimulatory effect by the BamHYSstI sitesof pXP2 (52). Zkansient Dansfections-For transfection experiments, HepG2 cells IL-6 or IFN-y, we introduced a clustered mutation in thispalindromic sequencewithin thecontext of the full-length ICA"1 were seededat 2 x lo5cells/well in 6-well plates (Costar), and 24 h later, promoter. We tested the inducibility of the mutant by transthe cells were transfected with 10 pg of supercoiled plasmidDNA by the calcium phosphate coprecipitation technique(531, consisting of a mixfecting HepG2 cells with this construct and measured luciferture of 6 pg of luciferase reporter plasmid, 4 pg of pSV-LacZ plasmid as ase activity after IL-6 or IFN-y treatment. As can be seen in a control plasmid to determine transfection efficiency. Following 16-20 Fig. 2 A , this mutation abolished the effects of both IL-6 and hexposure to the calcium-phosphateprecipitate,mediumwasreIFN-y on luciferase activity, demonstrating that this palinfreshed, and cells were incubated for 16 h with either IL-6(100 BSF-2 units/ml) or IFN--y (100 unitdml). Transfected cells were subsequently dromic sequence is necessary for maximal ICAM-1 gene transactivation by IL-6 and IFN-y. We have therefore designated harvested for luciferase assay (54) andlacZ determination (55). Gel Retardation Assays-Nuclear extracts were prepared from un- this region as a palindromic IL-GIIFN-y response element. To stimulated and stimulated HepG2 and U937 following cells a previously further test the IL-6 and IFN-y responsivenessof this pIRE, we described procedure (57).Oligonucleotides were labeledby filling in the inserted either one (1x1 or two (2x1 copies of a 23-bp synthetic cohesive ends with [CY-~~PI~ATPKlenow usingfragment of DNA polymerase I. Labeled DNA fragments were separated from unincorporated oligo containing the pIRE in front of the thymidine kinase vector and transfected these oligonucleotides by polyacrylamide gel electrophoresis.Gel retardation minimal promoter in the pl9tkluc assays were carried out accordingto published procedures with slight modifications (58). Briefly, nuclear extract (5 pg) was incubated in a

E. Caldenhoven, andP. Coffer, unpublished results.

I C A " 1 Promoter Regulation by IL-6 and IFN-y

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FIG.1. Functional analysis of the human ICA"1 promoter in HepG2 cells. A, schematic representationof potentialcis-acting elements in the human ICA"1 promoter, including the TATA boxes, TPA-responsiveelement, AP-2, NFKB,and pIRE site. The restriction sites shown are used for subcloning ICA"1 promoter fragments in frontof the luciferase (luc)gene of pXP2, A = AccI, B = BarnHI, H = HindIII, N = NheI, P = PstI, S = SrnaI, Ss = SstI. B , a series of 5' and internal deletions between -1014 and -37 of the ICA"1 gene were subcloned into the luc vector pXP2. HepG2 cells were transiently transfected by the calcium phosphate coprecipitation methodwith 6 pg of the luc reporter constructs and 4 pg of the P-galactosidase expression vector pSV-lacZ as a control for transfection efficiency.24 h after transfection cells were stimulated for 16 h with either IL-6 (100 BSF-2 unitshl) or IFN-y (100 unitshl). The luc activity for each construct was determined and corrected to p-galactosidase measured in the same sample. The values on the right show the -fold inductions of luc activity stimulated with IL-6 or IFN-y uersus untreated control cells. These values are averages of four independent experiments performed in duplicate, &.E.

constructs in HepG2 cells (Fig. 2B). We found that while the empty vector (without the pIRE enhancer elements) was not induced by IFN-y and only minimally by IL-6, activity of the lx pIREtkluc construct, with a single copy of the pIRE sequence was enhanced 2-fold by both IL-6 or IFN-y. The 2x pIREtkluc construct which contains two copies of this pIRE sequence showed a 5-fold induction by either IL-6 or IFN-y relative to tkluc alone.Taken together these results indicate that the stimulation of the ICAM-1 gene transcription by IL-6 and IFN-y is mediated through the same palindromic-responsive element. IL-6 and IFN-y Induce Nuclear Proteins, Which Specifically Bind to the ICA"1 pIRE-The PIRE-like element resides within the 70-bp upstream region (-105 to -37) that confers IL-6 and IFN-yselective inductionof the ICA"1promoter. We therefore examined in a gel retardation assay whetherspecific nuclear factors are induced by IL-6,or IFN-y which bind t o the ICA"1 pIRE. For this purpose we used a 32P-labeled oligonucleotide corresponding to the ICA"1 pIRE and nuclear extracts from HepG2 cells treated for increasing periods of time with IL-6 or IFN-y. As shown in Fig. 3A no pIRE binding activity was observed in nuclear extracts isolated from untreated HepG2 cells. Stimulation of HepG2 cells with IL-6 led to the induction of complex C1 (Fig. 3A), and this inducible almost binding activitywas maximal after 15min and returned to basal level after 1 h. Interestingly, we observed a complex retardation pattern suggesting that IL-6 might induce binding of more than one protein to the pIRE. Treatment with IFN-y also rapidly induced a nuclear protein binding to the pIRE; however, in contrast toIL-6, IFN-y induced only a single binding complex C2 which returned to basallevels after 24 h (Fig. 3B). To define binding specificity of the IL-6- and IFN-y-in-

duced complexes, competition experiments were performed in a gel retardation assay. Addition of an excess unlabeled pIRE(1C) completely inhibited the formation of the IL-6- and IFN-y-induced complexes (Fig. 3 C and D ) . As a control, competition with the mutant pIRE had no effect on the binding activity. We conclude from these resultsthat IL-6 and IFN-y rapidly induce distinct factors that specifically bind to theICAM-1 pIRE, however, with different dissociation kinetics. IFN-y and IL-6 Both Activate a GAF-related Factor That Binds to the ICAM-1pIRE, Whereas IL-6AlsoActivatesa DNAbinding Factor Related to APRF-Previously, it has been described that IFN-y activates a latent cytoplasmic 91-kDa DNAbinding protein, called the GAF (45). This p91 protein is also phosphorylated in response t o IFN-a and participates in the formation of theIFN-a-regulated,heterotetrameric protein complex termed ISGF3a (48, 58, 59). GAF binds to the GAS present in severalIFN-y-regulated promoters (43,461.We postulated that theIFN-y-induced pIRE-binding protein might be GAF and therefore tested a series of antisera against known IFN-related transcription factors, incubating them with nuclear extracts from HepG2 cells treated with IL-6 or IFN-y. Results show that theanti-91-kDa ISGF3a subunitantibody as well as anantibody that reactswith both, p91and p84 proteins (anti-p91/84) supershifted the pIRE complex C2 activated by antibody against the IFN-y effectively (Fig. 4A).In contrast, an 113-kDa subunit of ISGF3a did not reactwith either theIFN-yor IL-6-induced complexes. These results provide evidence that the DNA-binding complex induced by IFN-y contains a protein related to the 91-kDa subunit of ISGF3a which has recently been described as GAF.We further observed that the IL-6induced complex C2 is also recognized by the antibodies against p91 and p91/84. These results indicate that both IFN-)I and

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ICAM-1 Promoter Regulation by IL-6 and IFN-y A B

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FIG.2. Identification of a palindromicIL-6 and IFN-y-responsive element in the ICA"1promoter. HepG2 cells were transfected with the constructs detailed below as described under "Materials and Methods." 1day after transfection, cells werestimulated with either IL-6 or IFN-y and harvested after 16 h. The luciferase activity was normalized for the &galactosidase activity, and -fold induction by IL-6 or IFN-y was determined relative to the activity of untreated cells. On the left is a schematic representation of the chimeric constructs. Values on the right represent the averages of three independent experiments t S.E. A, site-directed mutagenesis of the pIRE in the ICA"1 promoter. The wild-type ICA"1 promoter (pIC-1014), or the mutant pIC-l014(pIREm) were transfected as above. B , The pIREsequenceconfersIL-6 and IFN-y responsiveness on a heterologous promoter. One( I x ) , or two ( Z x ) copies of a 24-bp oligonucleotidecontaining the pIRE from the ICA"1 gene was fused to the minimal thymidine kinase promoter-luciferase construct and transfected as above.

IL-6 induce the 91-kDa subunit of ISGF3a or a n antigenically cross-reactive complex. As previously mentioned the IL-6-induced C1-complex bears homology to theAPRF recently shown to bind to and transactivate the a,-macroglobulin promoter (36). To further investigate this similarity, we performed a gel retardation analysis utilizing the ICA"1 pIRE probe with IL-6-stimulated HepG2 cells and attempted to compete binding with an excess of unlabeled a,-macroglobulin APRF-binding site oligonucleotide (Fig. 4B). Indeed the cold probe effectively competed for complex C1 binding, further suggesting that this complex is related to the previously described APRF. IL-6 and IFN-y Induce Binding ofDistinctProtein Complexes to the ICA"1 pIRE in HepG2, Monocytic U937, and Differentiated U937 Cells-As already observed, IL-6 induces multiple complexes binding to the ICA"1 pIRE (Fig. 3 A ) . To obtain further information on the pIRE-binding complexes formed by IL-6 and IFN-y, we used nuclear extracts from HepG2 cells treated for different timeperiods with IL-6, IFN-y, or both and a better performed a "long-run" gel retardation assay to obtain separation of the complexes. As shown in Fig. 5 A , in nuclear protein extracts treatedfor 30 min with IL-6 three complexes C1, C2, and C3 were formed, whereas stimulation with IFN-y induced only the pIRE-binding complex C2. In contrast to induction by IFN-y, the IL-6-induced C2 complex was very transient, as it disappeared after 1h. Moreover, after treatmentfor 5 min withboth cytokinesa complex C3 was formed which may be due t o the formation of a heterodimer between the two

proteins involved in formation of the complexes C1 andC2 (see below). In a previous report we showed that ICA"1 expression is increased upon differentiation of U937 cells with TPA (17). It is further known that ICA"1 mRNA levels can be up-regulated by IL-6 and IFN-y in U937 cells? Therefore, we were interested if IL-6 and IFN-y induce similar transcription factors in these cells relative to those observed in HepG2 cells binding the I C A " 1 pIRE. For this purpose we used nuclear extracts from undifferentiated and TPA-differentiated U937 cells treated for 30 min with IL-6 or IFN-y and tested themfor binding to the pIRE of the ICAh4-1 gene. As shown in Fig. 5B,IL-6 or IFN-y treatment of undifferentiated U937 cells also induceda binding activity for this element which was comparable to that observed in HepG2 cells such that IL-6 also induced complexes C1, C2, and C3, while IFN-y induced only a C2-like complex. The C2 complex induced by either stimulus is clearly supershifted by p91-specific antisera demonstrating that it contains GAF or a n antigenically related protein. Furthermore, the IL6-induced C3 complex is also somewhat supershiftedby these antisera. TPA-induced differentiation of U937 cells abolished the induced binding activity by IL-6 to the ICA"1 pIRE, whereas IFN-y has an 2.3-fold enhanced induction of the C2like complex as determined by phosphoimager quantification (Molecular Dynamics). Interestingly, IFN-y appearedto induce a second complex of slightly lower mobility in these differentiE. Caldenhoven, and P. Coffer, unpublished results.

ICAM-1 Promoter Regulation by IL-6and IFN-y

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FIG.3. IL-6 and IFN-y induce nuclear factors inHepG2 cells that specifically bindto the ICA"1 pIRE. HepG2 cells were either untreated or treated with IL-6(100 BSF-2 unitdml) (A) or IFN-y (100 units/ml) ( B )for the indicated times. 5 pg of nuclear protein extract was mixed with the double-stranded "P-labeled ICA"1 pIRE oligonucleotide and analyzed by a gel retardation assay asdescribed under "Materials and Methods." Competition analysis was performed with 5 pg of nuclear protein extract from HepG2 cells treated for 30 min with IL-6 (100 BSF-2 unitdml) ( C )or IFN-y (100 unitdml) ( D ) .Nuclear extracts were incubated with the 32P-labeledICA"1 pIRE in the absence of competitor or in

ICAM-1 Promoter Regulation by IL-6and IFN-y

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FIG.4. Both IFW-y and IL-6 activates bindingof GAF to thepIRE, whereas IL-6also induces APRF. A, supershift analysisof ICAM-1 pIRE binding activity. Nuclear extracts were prepared from HepG2 cells treated for 15 min withIL-6 (100 BSF-2 units/ml) or IFN-y (100 unitslml). The extracts were incubated with either anti-p91, anti-p91/84, or anti-113 for antibodies 30 minbefore the additionof the "2P-labeled I C A " 1 pIRE. Gel retardation assay was performed as described in the legend toFig. 3 and under "Materials and Methods." B, competition analysis with the a,-macroglobulin APRE "core." Nuclear extracts were prepared from HepG2 cells treated for 30 min with IL-6 (100 BSF-2 unitslml). Nuclear extracts ( 5 pg of protein) were incubated with the 32P-labeled ICA"1 pIRE and were analyzed in a gel retardation As competitors assay. we used the indicated excess of unlabeled double-stranded oligonucleotides of the rat a2-macroglobulin APRE core site (a$ core), and the rat a,macroglobulin APRE mutatedcore site (mut core).

promoter can be stimulated by both IL-6 and IFN-y througha single regulatory element that appears to bind two distinct transcription factor complexes. Utilizing the human HepG2 hepatoma cell line we have demonstrated that the ICA"1 promoter is responsive to both IL-6 and IFN-y. The results presented were determined 16-h posttransfection but were also repeated after both 4 and 6 h, thus DISCUSSION reducing thepossibility of secondary factors being induced due Cell adhesion molecules such as ICA"1 are thought to play to exposure to cytokines for long time periods. The regulatory a role in immunological functioning by promoting cell-cell and element mediating thisresponsiveness has been localized to an cell-matrix interactions necessary for leukocytes to migrate to 11-bp motif 5'-"l'TCCGGGAAA-3' which we have termed the two the siteof inflammation (1).ICAM-1 can be expressedon a wide palindromic IL-6IIFN-y pIRE. Thiselementcontains variety of cell types and regulationof its expression is thought GGAA half-sites separatedby a single nucleotide and is similar to be an important mechanismof regulating inflammatory re- to that recently identified in other IL-6 and IFN-y-responsive sponses. Up-regulation of I C A " 1 expression by a variety of genes (19,42,Fig. 6). Althoughthe ICA"1pIRE contains some cytokines and inflammatory mediators such as IL-1, IFN-y, differences to the human GBP GAS sequence, it isvery homolTNF, and lipopolysaccharide has been reported, but mechathe ogous to other IFN-y-responsive gene elements such as the nisms underlying increased promoter activation are relatively human IRF-1 gene(Fig. 6). In fact theGAS sequence itself, the uncharacterized (5, 8, 60). We report here that the ICA"1 first p91GAF-bindingsite to be identified, appears to be one of

ated U937 derivatives, although we were unable to completely resolve this by gel retardation. These data indicate that IL-6 induces three complexes C1, C2, and C3 in both HepG2 and monocytic U937 cells and that IFN-y induces a pIRE-binding complex C2 in both undifferentiated and differentiated U937 cells similar to the one induced with IL-6.

the presence of 10 x, 50 x or 100 x molar excess (Mol. Ex. ) of unlabeled wild type I C A " 1 pIRE (pIRE[ICI) or mutant ICA"1 pIRE (pIRE[ml) oligonucleotide and analyzed by a gel retardation assay.

21152

ICAM-1 Promoter Regulation by IL-6 and IFN-y

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c3 b

c2 b

FIG.5.Analysis of the multiple complexes bind to the ICAM-1 pIRE in HepGP, U937, and differentiated U937 cells. A, nuclear extracts (5 pg of protein) fromHepGP cells were treated with I L 6 (100 BSF-2 unitdml), IFN-y (100 unitdml), or both cytokines for the indicated times and mixed with the labeled ICA"1 pIRE and analyzed in a long run gel retardation assay. B, nuclear extracts (5 pg) prepared from HepG2, U937, and TPA-differentiated U937 cells were either untreated or treated for 30 min with I L 6 (100 BSF-2 unitdml) or IFN-y (100 units/ml). Extracts were incubated with or without p9Y84 antisera and tested for binding to the 32P-labeled ICAM-1 pIRE and analyzed in a long run gel retardation assay.

B I IFN-y

IL-6 TPA

-

H ~. ~DGP

"

"

c1 b c3 b c2 b

A G C A T C G A TIT T A GIAIG T A AIT A T

hlCAM-I,plKE hIRF-I,IFN-yKE hGRP,GAS

FIG.6. Sequence comparison of the ICAM-I pIRE with other IL-6-and IFN-y-responsive binding sites. The palindromic structure of the ICA"1 pIRE is depicted in comparison with previously described IL-6- and IFN-y-responsive elements demonstrated to bind p91 or APRF. Sequences shown are from the rat a2-macroglobulin (rut d M ) ,human ICA"1 ( h l C M - I ) , human interferon regulatory factor-1 (hZRF-I),and human GBP gene (hGBP)promoters.

the lowest aftinity for p91. Mutations in key residues in the pIRE abolished responsiveness of the ICA"1promoter to both IL-6 and IFN-y demonstrating its importance in mediating cytokine responsiveness to these distinct hormones. Further-

I

+ " "

"

+

"

I

u937

+

+

+

-

+

- - - - - +

+

+

+

+

-

+

-

L1 " _

+

P 0

more, the pIRE element placed in thecontext of a heterologous promoter confers both IL-6 and IFN-y inducibility. The apparently similarspecificities of complexes binding to the pIRE does not imply that all IL-6-inducible sites are also responsive to IFN-y and vice versa. Indeed, we have found that the junB promoter contains an ICAM-like IRE that is not inducible by IFN-y5 This suggests that individual binding sites behave as IL-6 or IFN-y response elements or both, depending on the exact sequence structure. The similar response of the ICA"1 promoter to both IL-6 and IFN-y suggested that common factors were being induced by both IL-6 and IFN-y. To investigate the nature of these factors, we analyzed the pIRE binding activities in HepG2 cells treated with IL-6, IFN-y, and both cytokines together. Binding to the pIRE could be detected after only 5 min of cytokine P. Coffer, F. Horn, and W. Kruijer, manuscript in preparation.

ICAM-1 Promoter Regulation by IL-6 and IFN-y

21153

stimulation and reached maximum levels within 15 min. The blot analysis revealed the presence of GAF in both undifferencomplexes observed after treatment with IL-6 or IFN-y were tiated and monocytic U937 cells (66). The basis for the much overlapping but clearly distinct. Interestingly, these complexes higher levels of active GAF was proposed to be due to enhanced also had distinct time course of induction: the major IL-6-in- activating phosphorylation in thedifferentiated U937 cells. In GAF-related C2 duced C1 complex returned almost to basal levels after 60 min, contrast, we have demonstrated that the same while the IFN-y-induced C2 complex persisted for more than 20 complex appears tobe activated in undifferentiatedU937 cells in response to IFN-y stimulation as is present in HepG2 cells. h. This implies that distinct regulatory pathways are activated in response to stimulation by either IL-6 or IFN-y. Most inter- The discrepancy betweenthe results isdifficult to reconcile but estingly, IL-6 appears to be able to induce not only the C1 may be due to the utilizationof different bandshift conditions complex, but also two other complexes: the IFN-y-induced C2 and the higheraffinity I C A " 1 pIRE probe. The C2 complex is complex, and also a C3 complex. The converse is not true, in also activated by IL-6 in thesecells in a similar fashion to that that IFN-y is only capable of inducing the C2 complex. Analysis previously described in HepG2 cells. In theTPA-differentiated of a time course of induction in cells treated with IL-6, IFN-y, derivatives, however, there is a 2.3-fold enhancement of bindor both, provides a clue to the natureof the C3complex. After ing for the C2 complex and interestinglya complete abolition of 5 min of induction by IL-6 or IFN-7, only a single complex is IL-6 stimulation of the ICA"1 pIRE binding. The abolition of a down-regulation of observed in each case, complex C1 and complex C2, respec- IL-6-induced pIRE-binding may be due to tively. However, after 5 min of exposure t o a combination of IL-6 receptors on TPA-differentiated U937 cells. Recently, it has been shown that members of the Janus kiboth cytokines the third C3 complex is clearly observed. This suggests that thisC3 complex may be a heterodimer of the C1 nase family of protein tyrosine kinases are essentialfor intercomplex and C2 complex that canalso be formed by IL-6 treat- feron signaling (67, 68) and phosphorylation of ISGF-3a subment alone. Support of this is also seen in IL-6-treated U937 units (69,70).Indeed analysis of DNAbinding of ICA"1 pIRE cells where complex C3 is supershiftedby p91-specific antibod- and transactivationof the ICA"1 promoter by IL-6 and IFN-y ies (seealso below). Interestingly, the IL-6-induced C2 complex can be inhibitedby specific protein kinase inhibitors and actiis much more short-lived than its IFN-y-induced counterpart vation is Ras-inde~endent.~ This suggests that APRF not only suggesting that distinct regulatory pathways are activated by has similar binding properties to p91 GAF but also shares a similar activation pathway. Regulationof the ICA"1promoter the two cytokines. It is likely that the C2 complex is related to the previously may thus be tightly controlled not only by the availability of identified GAF factor as this complex can bespecifically super- cellular cytokine receptor types, but also the levels of various shifted by antibodies raised against GAF. Therefore GAF, or a ISGF3a family members present within a target cell. GAF-related protein complex, is likely to be the IFN-y-induced Acknowledgments-We thank Judith Johnson for the cDNAencoding transcription factor binding to the ICA"1 pIRE motif. Furthe ICA"1 gene, Chris Schindler for the kind gift of ISGF3 antibodies, thermore, preliminarycross-linking data demonstrate that the Ferdinand Vervordeldonk for photography, Gert Folkers for oligonucleC2 complex has a molecular mass of approximately 90 kDa6in otide synthesis, and members of the Hubrecht Laboratoryand Institute accordance with thatpreviously determined for GAF. It is clear for Biochemistry Aachen for helpful discussions. from the supershift data that the C1 and C2 complexes are Addendum-Following the submission of this article, a paper was different factors and that the C1 complex is distinct from GAF reported concerningthe cloning and characterizationof Stat3, a STAT as it cannot be supershifted by p91/84-specific antisera. The family member activated by tyrosine phosphorylation in response to activation kinetics comigration of complexes and competition EGF and IL-6(Zhong, Z., Wen, Z., and Darnell, J. E., Jr. (1994) Science experiments suggests that complex C1 is related to the previ- 264,95-98). This protein appears to be the previously described APRF ously described APRF (36). or complex C1 described above. Furthermore, it appears to form stable heterodimers with Stat1 ( ~ 9 1 resulting ) in a complex similar to C3 During the preparation of this article, several papers reported the activation of ISGF3a family members by diverse (described above). cytokines (61-64). It was observed that using a sis-inducible REFERENCES element a mobility shift containing threecomplexes (A, B, and 1. Springer, T. A. (1990)Nature 346.425433 C) could be observed after stimulating cells with IL-6 or EGF 2. Staunton, D. E., Marlin, S. D., Stratowa, C., Dustin, M. L., and SDringer, . - T.A. (1988) Cell 52,925-933 (64). In cells stimulated withEGF, it wasfound that antisera to 3. Diamond, M. S., Staunton, D. E., de Fougerolles,A. R., Stacker, S. A,, Garciap91 were able t o supershift complex B and C, while cells lackAguilar, J., Hibbs, M. L., and Springer, T. A. (1990) J. Cell. Biol. 111, ing p91 also lacked these two complexes (62, 64). Complex B 3129-3139 4. Duits, A. J., Dimjati, W., van de Winkel, J. G. J., and Capel, P. J. A. (1992) J. and C werethus postulated to contain (GAF) p91 while complex Leuk. Biol. 51, 237-243 A was not characterized. These complexes appear tocorrespond 5. Dustin, M. L., Rothlein, R., Bhan, A. K., Dinarello, C. A,, and Springer, T.A. (1986) J . Immunol. 137,245-254 t o the C1, C2, and C3 complexes described above and adds 6. Kvale, D., and Brandtzaeg, P. (1993) J. Hepatol. 17, 347-352 further support to the idea that complex C3 may be a het7. Mast, J., Schwaeble, W., Drach, J., Sommerauer, A,, and Dierich, M. P.(1992) erodimer of C1 and C2, e.g. p91 and APRF. J . Immunol. 148, 1635-1642 8. Rothlein, R., Czajkowski, M., ONeill, M. M., Marlin, S. 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Taga, T.,Hibi, M., Hirata, Y., Yamasaki, K., Yasukawa, K., Matsuda, T., Hirano, T., and Kishimoto, T.(1989) Cell 58, 573-581 aration of this article, the activation of GAF by IFN-y inU937 K., Lian-Jie, L., and Caughman, S. W. (1991) J . Biol. Chem. 266, cells was reported (66). In these experiments thepromonocytic 14. Degitz, 14024-14030 U937 cells exhibited virtually no activation of GAF binding t o 15. Stade, B. G., Messer, G., Riethmiiller, G., and Johnson, J. P. (1990) Immunobiology 182, 79-87 GAS probe after IFN-y addition, while in TPA-treated U937 16. Voraberger, G., Schafer, R., and Stratowa, C. (1991) J. Immunol. 147, 2777monocytes high levels of activated factor wereformed. Western 2786 E. Caldenhoven, and P. Coffer, unpublished results.

P. Coffer, E. Caldenhoven, and F. Horn, manuscript in preparation.

21154

I C A " 1 Promoter Regulation by IL-6 and IFN- y

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